Gs is critical for the actions of PTH and PTHrP. The gene encoding Gs (GNAS) also gives rise to XLs, which is expressed in various tissues including osteoblastic and renal cells. XLs can mimic Gs by stimulating cAMP generation in response PTH, although it is also predicted to have unique actions. Mutations in GNAS are found in several human diseases that impair signaling through the PTH/PTHrP receptor (PTHR). Most of these mutations affect both Gs and XLs. Studies in mice and humans indicate that XLs plays important roles in physiology and human disease, but the actions of XLs remain poorly understood. Our recent studies have provided novel insights into the cellular actions of XLs, and together with our findings obtained from XLs knockout (XLKO) mice, these led us to hypothesize that XLs is necessary for the regulation of calcium and phosphorus metabolism in vivo. In Aim 1 of the current proposal, we will address whether XLs is necessary for PTH-mediated actions in the renal proximal tubule during early postnatal development. We will determine a) whether proximal tubular actions of PTH in XLKO mice are impaired and b) whether the PTH resistance phenotype in XLKO mice is rescued by transgenically reconstituting XLs expression in the proximal tubule. In Aim 2, we will address whether XLs allows PTH actions to be sustained in the renal proximal tubule, which could explain the PTH resistance phenotype in XLKO mice. We will thus determine a) whether PTHR internalization is enhanced in the proximal tubule of XLKO mice; b) whether the PTH resistance phenotype in XLKO mice can be rescued by a mutant PTHR with sustained activity or by overexpressing Gs in the proximal tubule; and c) whether the interaction of XLs with dynamin influences PTH actions. These studies will provide novel insights into the actions of PTH and XLs in the renal proximal tubule, and these will be applicable to the actions of these proteins in skeletal tissues. Our results will also help reveal the roles of XLs in other systems, in addition to improving our knowledge of the mechanisms underlying the diseases caused by GNAS mutations. Given that XLs can activate the ubiquitous cAMP signaling pathway, our results will likely have even broader implications for human health and disease. PUBLIC HEALTH RELEVANCE: XLs is a variant of Gs, which is important for the actions of parathyroid hormone. Mutations in the gene making these two proteins cause different human diseases that affect the bone and mineral metabolism. Unlike Gs, functional roles of XLs are unknown. We aim to determine the role XLs in mediating the actions of parathyroid hormone to learn more about the mechanisms underlying those diseases.